The proposed research will develop methods of upregulating X-linked genes through technical innovations applied to the original RNA-activation technology developed in my laboratory (RNA-a; patent pending, PCT- US2011-065939). If successful, this next-generation RNA-a methodology will have the potential to cure dozens of intractable X-linked diseases such as Rett syndrome, Fragile X syndrome, and Duchenne muscular dystrophy. In the pharmaceutical industry, current therapeutic strategies focus almost exclusively on protein and microRNA targets. Yet, lncRNAs confer a temporal and spatial specificity not possible with proteins and small RNAs, and disease-associated mutations occur more often in noncoding than coding space. Therapeutic strategies might be as effectively targeted at long noncoding RNAs (lncRNA). Indeed, designing drugs against site-specific cis-acting lncRNAs would circumvent the pleiotropic effects plaguing modalities that general activities of epigenetic complexes via small molecules. The first-generation RNA-a strategy reawakens epigenetically silenced genes by disrupting binding between Polycomb repressive complex 2 (PRC2) and the lncRNA that targets it to the locus of interest. The RNA-a technology achieves the opposite of RNAi and has proven efficacious for various disease genes in preclinical testing. However, X-linked disease genes have not been amenable to RNA-a, likely because of many additional layers of heterochromatin resulting from X- chromosome inactivation (XCI). XCI is the mechanism of dosage compensation in mammals in which one X- chromosome is silenced in female cells to balance gene expression with male cells. My laboratory has specialized in the study of XCI for the past 16 years and recently discovered X-linked elements involved in the spreading of XCI along the inactive X chromosome (Xi). From these discoveries, we hypothesize that it should be possible to selectively reawaken specific Xi loci, without reactivating most or all of the Xi. Indeed, reactivating the entire Xi would be undesirable, as we have shown that global X-reactivation results in cancer. Thus, treating X-linked diseases requires a locus-specific approach to gene activation. With newfound knowledge of how XCI spreads, the envisioned next-generation RNA-a technology has the potential to do this. Because of our expertise in XCI and RNA-a, my lab is in a unique position to further develop the RNA-a technology to unlock expression of Xi genes in a locus-specific manner. Herein we propose to achieve this by leveraging gene-specific control elements along the Xi to reactivate the model disease gene, MECP2.